Interpretive Summary: Butyrate is produced by gut microbes and plays a critical role in energy metabolism and physiology in cattle. As a signaling molecule and a potent histone deacetylase inhibitor, butyrate functions as an anti-tumor and anti-inflammatory agent and has important implications in human health. However, its regulatory effect on alternative gene splicing has not been examined. This study detected numerous gene splicing events regulated by butyrate. Farmers may benefit from this study by developing means to selectively enhance butyrate synthesis in the rumen. Customers may benefit in the long term by having better and safer drugs designed to treat cancer and inflammation in the gut.

Technical Abstract:
As a signaling molecule and a potent inhibitor of histone deacetylases (HADCs), butyrate exerts its impacts on a broad range of biological processes, such as apoptosis and cell proliferation, in addition to its critical role in energy metabolism in ruminants. In this study, we examined the effect of butyrate on alternative splicing in the bovine epithelial cell using RNA-seq technology. Junction reads accounted for 11.28 and 12.32% of total mapped reads between the butyrate-treated (BT) and control groups (P<0.001). A total of 201,326 potential splicing junctions detected were supported by greater than or equal to 3 junction reads. Approximately 94% of these junctions conformed to the consensus sequence (GT/AG) while ~ 3% were GC/AG junctions. No AT/AC junctions were observed. A total of 2,834 exon-skipping events, supported by a minimum of greater than or equal to 3 junction reads, were detected. At least 7 genes, of which their mRNA expression significantly was affected by butyrate also had exon-skipping events differentially regulated by butyrate. COL5A3, which was induced 310-fold by butyrate (FDR <0.001) at the gene level, had significantly higher numbers of junction reads mapped to Exon#8 (Donor) and Exon#11 (Acceptor) in BT. This event had potential to result in the formation of a COL5A3 mRNA isoform missing 2 of the 69 exons. In addition, 216 differentially expressed transcript isoforms regulated by butyrate were detected. For example, Isoform 1 of ORC1 was strongly repressed by butyrate while Isoform 2 remained unchanged. Butyrate is a potent inhibitor of major HADCs. Our results suggest that butyrate also differentially regulated the expression of HDACs at the gene- and isoform- levels. In addition, 13 gene fusion events differentially affected by butyrate were identified. Our results provide a snapshot into complex transcriptome dynamics regulated by butyrate, which will facilitate our understanding of biological effects of butyrate and other HDAC inhibitors.